# Investigating the molecular requirements for ESX-1-lytic activity in pathogenic mycobacteria

> **NIH NIH R21** · UNIVERSITY OF NOTRE DAME · 2021 · $234,750

## Abstract

PROJECT SUMMARY
The mechanism of membrane lysis by the ESAT-6-system-1 (ESX-1) secretion system in pathogenic
mycobacteria remains elusive. The objective of this application is to define the ESX-1-associated lysin. The
hypothesis is that the conserved, pathogen-specific substrate, EspE promotes ESX-1 lytic activity by both M. tb
and M. marinum. To test this hypothesis, the following specific aims will be investigated. Under the first aim, the
applicant proposes to reroute the secretion of the EspE substrate and characterize the lytic activity and virulence
of the resulting strains. The objective of this aim is to define if the EspE substrate is necessary and sufficient for
ESX-1 lytic function and virulence. The working hypothesis is that rerouting EspE secretion will relieve the
requirement for the ESX-1 system in lysis and virulence. The hypothesis will be tested using molecular and
genetic approaches to bypass the requirement of ESX-1 for EspE secretion from M. marinum and M. tb and to
characterize the lytic activity and virulence of resulting strains. Under the second aim, the applicant proposes to
isolate and characterize nonlytic variants of the EspE substrate in M. marinum and M. tb. The objective of this
aim is to define how EspE promotes virulence in M. marinum and in M. tb. The working hypothesis is that EspE
is required for virulence because it promotes lysis. This hypothesis will be tested using molecular and
biochemical approaches to characterize the impact of the resulting EspE variants on ESX-1 mediated secretion
in vitro, lysis and virulence of both M. marinum and M. tb. The applicant expects that Aims 1 and 2 will contribute
seminal insight into how EspE and ESX-1 contribute to mycobacterial pathogenesis at the molecular level, and
demonstrate that EspE is functionally conserved in the human pathogen, M. tb. Moreover, completion of the
proposal will result in a pipeline to test if additional ESX-1 substrates function as lysins in pathogenic
mycobacteria. These contributions will be significant, moving the field vertically by drawing attention to substrates
other than EsxA and EsxB in understanding ESX-1-dependent lysis. This application is innovative because it
focuses on the previously understudied EspE substrate as a lysin. The experimental design is innovative
because it will produce a pipeline that can be used to test the requirement of any ESX-1 substrate for lysis and
virulence in pathogenic mycobacteria.

## Key facts

- **NIH application ID:** 10107976
- **Project number:** 1R21AI156229-01
- **Recipient organization:** UNIVERSITY OF NOTRE DAME
- **Principal Investigator:** Patricia A Champion
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $234,750
- **Award type:** 1
- **Project period:** 2021-03-19 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10107976

## Citation

> US National Institutes of Health, RePORTER application 10107976, Investigating the molecular requirements for ESX-1-lytic activity in pathogenic mycobacteria (1R21AI156229-01). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10107976. Licensed CC0.

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